Nonlinear optical imaging of extracellular matrix proteins

Chien Cheng Shih, Dennis M. Oakley, Matthew S. Joens, Robyn A. Roth, James A.J. Fitzpatrick

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

6 Scopus citations


Over the last 2 decades, nonlinear imaging methods such as multiharmonic imaging microscopy (MHIM) have become powerful approaches for the label-free visualization of biological structures. Multiharmonic signals are generated when an intense electromagnetic field propagates through a sample that either has a specific molecular orientation or exhibits certain physical properties. It can provide complementary morphological information when integrated with other nonlinear optical imaging techniques such as two-photon excitation (TPE). Here, we present the necessary methodology to implement an integrated approach for multiharmonic and TPE imaging of the mouse aorta using a commercial two-photon microscope. This approach illustrates how to differentiate the microstructure of the mouse aorta that are due to collagen fibrils and elastic laminae under 820 and 1230 nm excitation. Our method also demonstrates how to perform multiharmonic generation by reflectance of the forwardly propagating emission signal. The ability to visualize biological samples without additional genetically targeted or chemical stains makes MHIM well suited for studying the morphology of the mouse aorta and has the potential to be applied to other collagen and elastin-rich tissues.

Original languageEnglish
Title of host publicationMethods in Cell Biology
PublisherAcademic Press Inc.
Number of pages22
StatePublished - 2018

Publication series

NameMethods in Cell Biology
ISSN (Print)0091-679X


  • Collagen
  • Elastin
  • Mouse aorta
  • Multiharmonic generation
  • Nonlinear optical microscopy
  • Second-harmonic generation
  • Third-harmonic generation
  • Two-photon excited fluorescence


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